NK-1 is one of five homeobox genes clustered in the 93E1-5 region of the third chromosome of Drosophila (NK-1, NK-3, NK-4, Nkch4 and 93Bal; Kim and Nirenberg, PNAS 86: 7716, 1989; Jagla et al., Gene 127: 165, 1993). The expression pattern of NK-1 revealed by in situ hybridization showed that specific muscle segments and a subset of neuronal cells in the ventral nerve cord expressed NK-1, implying that NK-1 may be regulated in a tissue-specific manner and that NK-1 may be involved in muscle segment formation and/or neuro-muscular synaptogenesis. To determine the cis-acting DNA elements controlling the tissue-specific expression of NK- 1, we measured transiently expressed chloramphenicol acetyltransferase (CAT) activity from transfected C2C12 myoblasts and NG108-15 neuroblastoma cells using various CAT constructs containing different 5' upstream regions of NK-1. From the analysis of 4 kb of the 5' upstream region, it was shown that the E5 region (676 bp) had strong enhancer elements. Mutant analysis of this region showed that an 86 bp DNA fragment (-435 to -350) was necessary and sufficient to demonstrate enhancer activity in C2C12 cells. However, additional regions (-157 to - 28 and -510 to -425) were required for optimal enhancer activity in NG108-15 cells. Gel-shift assays and Dnase I footprinting assays defined a known binding site for NF-IL6, 5'-TTTCGCAAG-3' (-425 to -417) and a novel binding site, 5' AATTACTCACATCC 3' (-370 to -357), for unknown factors. We have also identified a strong silencer region (-1865 to - 476). This negative element functions both in myoblast and neuroblastoma cells. The results suggest that the cell-specific expression of the NK-1 homeobox gene in transfected C2C12 myoblasts and NG108-15 neuroblastoma cells may be regulated through both silencer and multiple enhancer DNA elements. We are also continuing to screen mutant flies for NK-1 using the enhancer trap method. We have cloned and sequenced a novel mouse homeobox gene, Nkx-1, which is a homologue of Drosophila NK-1. We have also constructed plasmids containing Nkx-1 DNA disrupted by PGK-neo for the purpose of knocking out the Nkx-1 gene in embryonic stem cells by homologous recombination.